Fig 1: S1P induced IL-1β and IL-18 secretion in NLRP3 inflammasome-dependent manner. (A) Representative images of immunofluorescence analysis by confocal microscopy in serum-starved BMMs treated with 1 μmol/L S1P for 6 h. White arrows indicate oligomerized ASC. (B) The relative expression of NLR and non-NLR PRR family members were examined by qRT-PCR in serum-starved BMMs treated with S1P for 2 h. (C) ELISA measurements of IL-1β and IL-18 in culture supernatants from serum-starved BMMs pretreated with 100 nmol/L MCC950 for 1 h, followed by treatment with 1 μmol/L S1P for 12 h, or 100 ng/ml LPS for 4 h, then 5 mmol/L ATP for 30 min. Data are presented as the mean ± SEM. n = 6 per group. Student's t-test was used in panel (B) and one-way ANOVA was used in panel (C). *p < 0.05 vs. control. #p < 0.05 vs. S1P treatment alone. &p < 0.05 vs. LPS plus ATP treatment.
Fig 2: Effects of Z. morio hemolymph on E. coli-induced inflammatory response in PMECs. (A) The percentage of E. coli adhesions detected by adhesion assay of bacteria with PMECs. (B) CFU detection of E. coli in supernatants. (C) CCK-8 assay analysis of cell death of PMECs in each group at 12 h after E. coli infection. (D–G) The mRNA expression of IL-1β, IL-18, IL-6 and Tnf-α in PMECs at 6, 12, 24 h post-infection. XTRC beetle stands for Z. morio hemolymph-treatment. Data presented are means ± SEM (n = 3 per group). * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig 3: 3-MA reverses the protective effect of hydrogen sulfide on LPS-induced acute kidney injury in mice by inhibiting autophagy. (A) The expression of LC3B and p62 proteins in mouse kidney tissues after 3-MA treatment. (B) The ratio of p62/β-actin. (C) The ratio of LC3B-II/LC3B-I. (D) Immunofluorescence analysis of LC3B punctate aggregation in kidney tissue. Scale bar, 20 µm. The red arrows indicate autophagosome. (E) The BUN level in mouse serum after 3-MA treatment. (F) The creatine level in mouse serum after 3-MA treatment. (G) The morphological changes of kidney tissue under light microscope after 3-MA treatment (hematoxylin and eosin staining). Scale bar, 20 µm. (H) Histological damage score of mouse kidney. *P<0.01 vs. control group; **P<0.01 vs. LPS group; ***P<0.01 vs. LPS + NaHS group. (I) IL-1β and (J) IL-18 levels in mouse serum after 3-MA treatment. (K) IL-1β and (L) IL-18 levels in kidney tissue after 3-MA treatment. (M) TUNEL staining shows the level of apoptosis in kidney tissue after 3-MA treatment. Scale bar, 20 µm. (N) Statistics of apoptosis index in mouse kidney tissue of each group. Data are presented as the mean ± SD (n=10). 3-MA, 3-methyladenine; LPS, lipopolysaccharide; LC3B, microtubule associated protein 1 light chain 3; IL, interleukin; BUN, blood urea nitrogen; NaHS, sodium hydrosulfide hydrate.
Fig 4: The expression of NLRP3 inflammasome-related genes in serum of patients with clinical sepsis. (A–D) The expression of IL-1β, IL-18, TRAF3, and SGT1 in serum of patients with clinical sepsis was detected by ELISA. Data are representative or means (SD) of at least three independent experiments. Statistical analyses were determined using a Student’s t-test.
Fig 5: NU9056 inhibited apoptosis and inflammation of microglia in vitro. (A) Apoptosis rate in each group was detected using flow cytometry. (B) The average fluorescence intensity of each group was measured using flow cytometry. (C) Cell viability was tested using CCK8 assay. (D) The IL-1β and IL-18 release were estimated using ELISA. *P < 0.05.
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